CN117511826B

CN117511826B - Lactobacillus mucilaginosus and application thereof

Info

Publication number: CN117511826B
Application number: CN202410000615.8A
Authority: CN
Inventors: 曾婉秋; 王琼; 高翔
Original assignee: Sichuan Anaerobic Biotechnology Co ltd
Current assignee: Sichuan Anaerobic Biotechnology Co ltd
Priority date: 2024-01-02
Filing date: 2024-01-02
Publication date: 2024-03-15
Anticipated expiration: 2044-01-02
Also published as: CN117511826A

Abstract

The invention relates to the field of microorganisms, in particular to lactobacillus salivarius and application thereof. The lactobacillus casei has no virulence factor, no drug resistance gene, no hemolysis and good safety. The bacterium has strong lactic acid production capability, can reduce female vagina pH, and inhibit pathogenic bacteria reproduction. In addition, antibacterial substances such as hydrogen peroxide, lactic acid, bacteriocin and the like generated by the gardnerella vaginalis have better antibacterial capability.

Description

Lactobacillus mucilaginosus and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to lactobacillus salivarius and application thereof.

Background

The female genital tract is an open cavity tract, is lodged with a large number of different kinds of microorganisms, is one of main distribution areas of human microorganisms, and is closely related to female reproductive health. About 300 or more microorganisms are symbiotic in the female genital tract, including bacteria, viruses, fungi, etc., with bacteria being the predominant microorganism. They are constrained and balanced with each other to form dynamic balance, and various vaginitis is related to the imbalance of the vaginal microecological environment.

The microbial composition of the genital tract varies from site to site, and in healthy women of childbearing age most bacteria are present in the lower genital tract (vagina and cervix) and the bacteria in the upper genital tract have not been well characterized to date. The vaginal microbiota has low microbial diversity, and is mainly composed of lactobacillus. The vaginal flora can be generally divided into five community (community state types, CST) types. CST-I is mainly composed of Lactobacillus crispatus, CST-II is mainly composed of Lactobacillus gasseri, CST-III is mainly composed of Lactobacillus jensenii, CST-V is mainly composed of Lactobacillus jensenii, and CST-IV has no dominant Lactobacillus group and can be further subdivided into CST IV-A and CST IV-B subtypes, wherein CST IV-B is closely related to bacterial vaginosis (Bacterial Vaginosis, hereinafter referred to as "BV") and mainly composed of anaerobic bacteria such as Bacillus mirabilis, goldebrand bacteria and Gardnerella.

Under the disease state, the microorganism composition and the biomass load change greatly, a plurality of pathogens overgrow, the vaginal microecological environment enters a fragile state, and the infection and the reproduction of the pathogens are not easy to resist, and various vaginal inflammations appear. BV is a common gynecological disease, the infection rate is 15% -52%, and is a vaginal infectious disease with clinical syndrome caused by dysbacteriosis in vagina due to excessive reproduction of gardnerella vaginalis and other anaerobic bacteria to replace lactobacillus. BV is reported to be a risk factor for histological choriitis, amniotic fluid infection, post-cesarean endometritis, and other pregnancy defects and pregnancy complications.

Aiming at BV, the clinical treatment method adopts the antibiotics metronidazole or clindamycin, the metronidazole is a precursor, and in an anaerobic environment, the nitro of the metronidazole is reduced to amino by the intracellular enzymatic reduction of bacteria, so that the antibiotics are converted into an active form, and then the helix structure of the antibiotics is destroyed by covalent bonding with pathogen DNA, single strand and double strand breaks are caused, so that the pathogen DNA is degraded and dead; clindamycin can bind to the 50S ribosomal subunit on the bacterial ribosome, preventing extension of the peptide chain, thereby inhibiting protein synthesis by bacterial cells, resulting in bacterial death. The treatment with antibiotics is quick in effect, but has great defects, and has the following two aspects: (1) The vaginal microecology is not restored to a healthy equilibrium state which can resist pathogenic bacteria invasion after treatment, and the inhibited or killed pathogenic microorganisms or external pathogenic microorganisms can be further propagated and even pathogenic to cause recurrence or new vaginal inflammation; (2) The microorganisms produce drug resistance, and antibiotics cannot balance the vaginal microecological environment, resulting in refractory BV. Thus, although the antibiotic treatment takes effect quickly, the recurrence rate is high, and the recurrence rate is up to 30% within 3 months.

The treatment of the vaginal microecological imbalance comprises three steps of sterilization, mucous membrane repair and restoration of vaginal microecological balance. Sterilization is the first step in the treatment of vaginal inflammation, and inhibits or kills pathogenic microorganisms, including hyperproliferative aerobic and anaerobic bacteria, blastospores or fungi, trichomonas, and the like. After the pathogenic microorganisms are inhibited or killed, the immune repair of the vaginal mucosa and the recovery of dominant lactobacillus are the final targets for treating the colpitis. During this period, if the repair of the vaginal mucosa, the recovery process of lactobacillus is affected, the physical and chemical environment in the vagina is not restored to normal, the suppressed pathogenic microorganisms or the external pathogenic microorganisms can also reproduce again or even cause diseases to cause recurrence or new vaginal inflammation. And probiotics can rapidly occupy the receptors of vaginal epithelium in the vagina to generate a protective effect on the vagina, thereby promoting the vagina to restore to the normal microenvironment and reducing the recurrence of colpitis. Therefore, the probiotic microecological preparation is the most preferable mode for treating BV in the current technical means.

At present, only 2 vaginal microecological medicines are marketed in China, and one of the medicines is streptococcus enteritidisStreptococcus faecalis) The other is Lactobacillus delbrueckiiLactobacillus delbrueckii) Is active ingredient. In addition, some drug lines are in the stage of clinical development, such as the European micro-family of live bacteria capsules containing Lactobacillus crispatus Lc262-1, which has recently been subjected to phase 3 clinical trials, and KAL-001 live bacteria capsules containing 4 dominant lactobacilli from Sichuan anaerobic organisms are in phase 2 research. In addition to pharmaceuticals, some oral probiotics have emerged in the art, such as UREX developed by kehansen ^® And ASTART E ^® Has been widely used in various applications for female reproductionIn oral products for health problems, the principle is reported to be the transmission of probiotics to the vagina via the oral-intestinal-anal route.

Chinese patent document CN 102851248A discloses a lactobacillus jensenii for the control of bacterial vaginosis. Chinese patent document CN 107794236A discloses a lactobacillus crispatus and its use. Chinese patent document CN 108004187A discloses a lactobacillus gasseri and its use in the preparation of a medicament for inhibiting bacteria in the vagina.

Because the human body has rich lactobacilli in vagina, different lactobacilli show different probiotics, the different lactobacilli play a role in cooperation in the vaginal microenvironment, and individual differences exist, and dominant bacterial strains in different female vagina are slightly different, the types of the lactobacilli and the probiotics of different bacterial species need to be comprehensively considered when the corresponding lactobacilli probiotics for vagina are selected.

Lactobacillus salivarius isolated from urine of healthy females was first reported in 2021 and its beneficial and related effects on humans have not been reported. The lactobacillus salivarius strain provided by the invention is separated from healthy female vaginal secretion, has good safety, has good probiotic functions in the aspects of acid production, hydrogen peroxide, bacteriostasis and the like, and is beneficial to solving the problem of female genital tract health.

Disclosure of Invention

The first aim of the invention is to provide a lactobacillus mucilaginosusLimosilactobacillus urinaemulieris) The strain is selected from lactobacillus helveticus Lvagi-34 with a preservation number of CCTCC NO: M20231491.

In some embodiments, the strain has a 16S rDNA sequence as set forth in SEQ ID NO. 1.

The second object of the present invention is to provide a method for culturing the aforementioned strain of lactobacillus casei, comprising inoculating the strain of lactobacillus casei into a medium and culturing the same in a proliferation manner to obtain a proliferated strain of lactobacillus casei.

In some embodiments, the medium is MRS medium.

The third object of the present invention is to provide a food, health product or pharmaceutical composition comprising the aforementioned Lactobacillus salivarius strain or the Lactobacillus salivarius strain obtained by the aforementioned culture method as an active ingredient.

In some embodiments, the active ingredient of the composition further comprises one or more selected from the group consisting of lactobacillus crispatus, lactobacillus jensenii, lactobacillus johnsonii, lactobacillus delbrueckii, and lactobacillus grignard.

In some embodiments, the lactobacillus salivarius strain is the sole active ingredient.

In some embodiments, the composition comprises 10 in a single formulation ⁶ ~10 ¹⁵ Urinary tract mucus lactobacillus of CFULimosilactobacillus urinaemulieris) Strains.

A fourth object of the present invention is to provide the use of the aforementioned strain of lactobacillus mucilaginosus for the preparation of a product for improving the health of the female genital tract.

In some embodiments, the product that improves female genital tract health is used as a bacteriostatic or bacteriocidal agent.

In some embodiments, the product for improving female genital tract health is used to inhibit or kill gardnerella vaginalis.

In some embodiments, the Lactobacillus salivarius Lvagi-34 of the present invention has the following morphological characteristics:

(1) Microscopic examination of the strain to gram-positive Brevibacterium;

(2) The colony form in MRS solid culture medium is light white semitransparent circular colony, the middle is convex, and the surface is smooth and moist.

The lactobacillus salivarius Lvagi-34 has no virulence factor, no drug resistance gene and no hemolysis, and has good safety. The lactic acid producing capability is strong, the female vagina pH can be reduced, and the bacteriostatic substance hydrogen peroxide can be produced, so that the pathogenic bacteria reproduction can be effectively inhibited.

The strain preservation information provided by the invention is as follows:

strain name: lactobacillus mucilaginosus in urinary tractLimosilactobacillus urinaemulieris）Lvagi-34

Preservation date: 2023, 08, 17

Preservation unit: china center for type culture collection (China Center for Type Culture Collection, CCTCC), address: university of martial arts, hubei province, post code: 430072, telephone: 027-68754052

Preservation number: cctccc No. M20231491.

Drawings

FIG. 1 is a front view of the colony morphology of Lactobacillus salivarius Lvagi-34 in example 1.

FIG. 2 is a gram of Lactobacillus salivarius Lvagi-34 of example 1.

Detailed Description

Definition and description

Lactobacillus salivarius strains of specific accession numbers as claimed in the present invention, the meaning of which includes, but is not limited to:

lactobacillus casei of urinary tract with CCTCC NO: M20231491 as the deposited microorganismLimosilactobacillus urinaemulieris) Lvagi-34 strain;

a strain of lactobacillus casei having the same genome as the lvagei-34 strain;

the passaging strain without mutation based on the aforementioned 1 or 2;

a passaging strain based on the aforementioned 1, 2 or 3 that accumulates minute mutations in passaging, but has no substantial change in toxicity, immunogenicity and biological activity;

based on the live or inactivated form of the strain according to any of the foregoing 1-4, it may be whole cells or derivatives such as lysates or fermentation products.

As known in the art, it is reasonable to expect that minor mutations are unavoidable when the mutation occurs in a non-coding sequence region or synonymous mutation of the coding region or mutation that does not affect the toxicity, immunogenicity and biological activity of the strain (e.g., a linked amino acid residue that may be located between two domains or a residue that is located within the higher structure of the protein and does not affect toxicity, immunogenicity and biological activity by not contacting immune cells), and that the objective of the invention can still be achieved without such minor changes significantly affecting toxicity, immunogenicity and biological activity of the progeny strain, and that it is derived from the strain contributed by the invention and therefore still falls within the substantial technical contribution of the invention. These minor mutations remain insubstantial mutations and should be considered as mutant strains that have no alterations in toxicity, immunogenicity, and biological activity.

There is no substantial change in toxicity, immunogenicity, and biological activity, including, but not limited to, regarding toxicity, immunogenicity, and biological activity as being the same within the limitations and acceptable or unavoidable errors of detection techniques such as detection sensitivity, detection limits, and the like. The toxicity, immunogenicity and biological activity of the L vagi-34 strain offspring were determined by cells, animals, etc., and the expected or unavoidable systematic errors were attributed to insubstantial changes due to differences in cell lines, animal varieties, age, sex, health conditions, culture conditions, etc.

The composition contains an active ingredient Lvagi-34 strain and other ingredients, such as auxiliary ingredients without physiological effects or other functional ingredients. Functional ingredients include, but are not limited to, other functional strains, or prebiotics, metazoan ingredients, and the like.

As a preferred mode, the composition of the invention contains other active lactobacilli, such as one or more selected from the group consisting of Lactobacillus crispatus, lactobacillus jensenii, lactobacillus johnsonii, lactobacillus delbrueckii and Lactobacillus grignard.

The auxiliary materials comprise additives, drug carriers and excipients. A pharmaceutical carrier refers to a pharmaceutical carrier that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered probiotic. The pharmaceutically acceptable carrier may enhance or stabilize the composition or may be used to facilitate the preparation of the composition. Pharmaceutically acceptable carriers can include solvents, dispersion media, coatings, surfactants, antioxidants, isotonic agents, absorption delaying agents, salts, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., remington's Pharmaceutical Sciences, 18 th edition MackPrinting Company,1990, pages 1289-1329). Unless the conventional carrier is incompatible with the active ingredient, it is contemplated that it will be used in a therapeutic or pharmaceutical composition. The carrier may be selected to minimize adverse side effects in the subject and/or minimize inactivation of the active ingredient.

An excipient refers to a substance that is added to a pharmaceutical composition to give the drug a certain shape or a certain concentration. Such as sterile water, physiological saline, polyalkylene glycols (such as polyethylene glycol), vegetable oils or hydrogenated naphthalenes, calcium bicarbonate, calcium phosphate, various sugars, various types of starch, cellulose derivatives, gelatin, and the like.

The composition of the present invention may be prepared in any form convenient for use, such as powder, tablet, granule, gel, capsule or liquid, which are common in clinical or food.

The compositions of the present invention are administered to a subject in an amount (therapeutically effective amount) and frequency effective to exert efficacy, preferably in a single dose of 10 ⁶ ~10 ¹⁵ CFU、10 ⁷ ~10 ¹³ CFU or 10 ⁷ ~10 ¹² Urinary tract mucus lactobacillus of CFULimosilactobacillus urinaemulieris）。

The specific temperature parameters in the present invention, unless specified otherwise, are understood to be constant temperature treatments and allow for variations within a certain temperature interval. Such as within a range of + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.

The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and that all other embodiments obtained by a person skilled in the art without making creative efforts based on the embodiments in the present invention shall fall within the protection scope of the present invention.

The materials used in the following examples were formulated or purchased as follows:

MRS broth preparation: MRS finished product culture medium (Ther)mo Scientific ™, CM 0359B) powder 52.0 g, dissolved in 1L distilled water; heating to boil, cooling to room temperature, adding 0.55 g cysteine hydrochloride, stirring to dissolve, and adjusting pH to 6.5; installing quantitative liquid separator and introducing N ₂ Heating to boil, boiling for 20 min, cooling, packaging into 10 mL anaerobic tube, sterilizing at 118 deg.C under moist heat for 20 min, storing in shade and in dark place.

MRS solid culture medium preparation: weighing MRS finished product culture medium (Thermo Scientific ™, CM 0359B) powder 52.0 g and agar powder 15.0 g, dissolving into distilled water 1L, boiling, adding cysteine hydrochloride 0.55 g after boiling, adjusting pH to 6.5, sterilizing at 118 deg.C under moist heat for 20 min, and storing in shade and in dark for use.

Hydrogen peroxide semi-quantitative medium preparation: weighing MRS finished product culture medium (Thermo Scientific ™, CM 0359B) powder 52.0 g, agar powder 15.0 g, dissolving in 1L distilled water, adjusting pH to 6.5, sterilizing at 118 deg.C under high temperature and humidity for 20 min, placing into a 50deg.C water bath after sterilization, maintaining for 30 min, adding 3,3', 5' -tetramethyl benzidine (TMB) (final concentration is 0.25 mg/mL) and horseradish peroxidase (HRP) (final concentration is 0.01 mg/mL), and mixing; cooling and solidifying, and placing in a refrigerator at 4 ℃ for standby.

Preparation of anaerobic PBS: weighing potassium dihydrogen phosphate 0.27 and g, disodium hydrogen phosphate 1.42 and g, sodium chloride 8 and g, and potassium chloride 0.2 and g, dissolving in distilled water 1 and L, heating and boiling, cooling to room temperature, adding cysteine hydrochloride 0.55 and g, stirring and dissolving, adjusting pH to 6.5, loading into quantitative liquid separator, and introducing N ₂ Heating to boil, boiling for 30 min, cooling, packaging into 10 mL anaerobic tube, sterilizing at 121deg.C under moist heat for 30 min, and storing in shade and in dark place.

Preparing an anaerobic BHI liquid culture medium: weighing BHI product culture medium (Thermo Scientific ™, CM 1135B) powder 37.0 g, dissolving in distilled water 1L, heating to boil, cooling to room temperature, adding cysteine hydrochloride 0.55 g, stirring to dissolve, adjusting pH to 6.5, loading into quantitative dispenser, and introducing N ₂ Heating to boil, boiling for 20 min, cooling, and packaging with N ₂ And CO ₂ Packaging into 10 mL anaerobic tubes at a ratio of 1:1, sterilizing at 118 deg.C under moist heat for 20 min, and storing in shade and in dark for use.

Preparing an anaerobic BHI semi-solid culture medium: weighing BHI finished medium (Thermo Scientific ™, CM 1135B) powder 37.0 g, dissolving into 1L distilled water; heating to boil, cooling to room temperature, adding 6 g agar powder, 0.55 g cysteine hydrochloride, stirring to dissolve, adjusting pH to 6.5, adding quantitative liquid separator, and introducing N ₂ Heating to boil, boiling for 20 min under slight boiling condition, slightly cooling, and packaging with N ₂ And CO ₂ Packaging into 10 mL anaerobic tubes at a ratio of 1:1, sterilizing at 118 deg.C under moist heat for 20 min, and storing in shade and in dark place.

Example 1 isolation and identification of strains

Collecting vaginal secretion samples of 20-40 year old Chinese healthy women by using a vaginal cotton swab, taking 2 mL sterile and anaerobic PBS buffer solution in an anaerobic tube filled with the cotton swab, fully vibrating and uniformly mixing, and continuously carrying out ten-fold gradient dilution by taking the solution as a stock solution; coating 100 mu L of liquid diluted 10000 times on an MRS solid culture medium, placing the solid culture medium in an anaerobic incubator for culture at 37 ℃, after culturing 48 and h, selecting a plurality of single colonies to respectively culture 24 h in an MRS broth culture medium, transferring one part of the cultured bacterial liquid for continuous culture, and extracting bacterial DNA from the other part of the bacterial liquid; amplifying and sequencing bacterial 16S rRNA gene, and BLAST comparing the sequencing result to determine 3 strains of urinary tract mucus lactobacillusLimosilactobacillus urinaemulieris) Named as Lvagi-34, lvagi-35 and Lvagi-42 respectively.

And (3) taking lactobacillus mucilaginosus Lvagi-34 strain, and marking the strain liquid to MRS solid culture medium after PCR verification, and carrying out anaerobic static culture at 37 ℃ for 24-48 h, wherein the colony form is light white semitransparent circular colony, the middle part is convex, the surface is smooth and moist, and the front photograph is shown in figure 1.

1-loop urinary tract mucus lactobacillus Lvagi-34 is taken to be dropped on a glass slide, and a proper amount of sterile ddH is added dropwise ₂ O is coated as a thin bacterial liquid layer, and the glass slide is placed on an alcohol lamp and heated until water evaporates, and the glass slide passes through the flame for 2-3 times rapidly to fix the bacterial cells. Along with itThe staining was then performed according to the gram stain kit instructions (Guangdong CycloKai microorganism technologies Co., ltd., 029010). Microscopic examination, observation and photographing, and the bacterial staining mirror is used for detecting gram-positive Brevibacterium, and the gram-positive Brevibacterium is shown in FIG. 2.

EXAMPLE 2 genome-wide novel analysis and preservation of strains

Lactobacillus salivarius Lvagi-34 was inoculated into 5 ml MRS broth medium, cultured to logarithmic growth phase, and then strain whole genome DNA was extracted and subjected to whole genome sequencing using Illumina high throughput sequencing platform Novageq 6000. After assembly and annotation by conventional methods, protein sequences were entered into VFDB (Virulence Factor Databases) and CARD (The Comprehensive Antibiotic Resistance Database) databases for virulence factor and drug resistance gene analysis, respectively. The results show that the bacterium does not have virulence factors and drug resistance genes.

The novel analysis of the strain was performed using the average nucleotide similarity (Average Nucleotide Identity, ANI). By searching in Genbank, 5 published results were foundLimosilactobacillus urinaemulierisWhole genome, found by fastANI (v 1.33) comparison, 2 strains were closest to the Lvagi-34 whole genome and below 99.9%, GCA_014838745.1 (98.8398%) and GCA_932750685.1 (97.5919%), respectively. Thus, lvagi-34 is considered to be a novel strain, and the 16S rDNA sequence thereof is shown in SEQ ID NO. 1.

Example 3 Low pH growth tolerance experiment

The preserved Lactobacillus urinary tract mucus Lvagi-34 was activated in MRS broth pH 6.5 and incubated overnight at 37 ℃. Transferring the activated bacteria liquid into MRS broth culture medium with pH value of 4-5 at a ratio of 10%, and measuring OD every 2-3 h ₆₀₀ Values.

The results show that Lactobacillus salivarius Lvagi-34 can grow in a low pH environment (pH 4-5) and has acid-resistant properties.

Example 4 lactic acid production test

Respectively activating urinary tract mucus lactobacillus Lvagi-34, lvagi-35 and Lvagi-42, transferring into MRS broth culture medium, culturing at 37deg.C for 48 h in 2 parallel strains, measuring with pH 0.5-5.0 test paper, recording pH value of lactobacillus strain liquid after culturing for 48 h, and selecting strain for liquid chromatography according to the following two conditions. The supernatant was diluted 5-fold, pretreated with concentrated sulfuric acid, and filtered through a 0.22 μm needle filter before loading. The liquid chromatography related parameters are as follows:

instrument model: agilent, analytical liquid chromatography 1200

Chromatographic column model: bere, aminex HPX-87H

Mobile phase: 0.005 M H ₂ SO ₄ At a speed of 0.6 mL/min

Detector and detection wavelength: DAD,207 nm; RID, differential refractive signal

Sample injection amount: 20. mu L.

The results are shown in Table 1, in which Lactobacillus salivarius Lvagi-34 produces lactic acid to lower pH. Under the same experimental conditions, the lactic acid producing capacity of Lvagi-34 is stronger than that of the control strains Lvagi-35 and Lvagi-42 of the same genus.

Table 13 bead Lactobacillus urinary tract mucus lactic acid producing ability and pH value of culture for 48 h

Microorganism	Average lactic acid content (mg/L)	Average pH (48 h)
			Lactobacillus salivarius Lvagi-34	13369.9780	3
Lactobacillus salivarius Lvagi-35	11797.2532	3.5
			Lactobacillus salivarius Lvagi-42	10413.3975	3.5

EXAMPLE 5 Hydrogen peroxide production capability

After the urinary tract mucus lactobacillus Lvagi-34, lvagi-35 and Lvagi-42 are activated, 2 mu L of bacteria liquid is inoculated in MRS solid culture medium containing 0.25 mg/mL TMB and 0.01 mg/mL HRP by a liquid transfer device, a plate is placed in the same anaerobic sealed tank, an anaerobic gas generating bag is added for culture at 37 ℃, at 48 h observation time points, the corresponding plate is taken out and exposed to air, and after 30 min, the color reaction is observed and photographed for recording: the blue color produced by Lactobacillus delbrueckii was 4 points, the blue color produced by Lactobacillus delbrueckii was 3 points, the blue color produced by Lactobacillus delbrueckii was 2 points, the blue color was very weak, and the color was 1 point (slight color reaction) and the color was not changed, respectively, 0 points.

As a result, as shown in Table 2, lactobacillus salivarius Lvagi-34 produced a certain amount of hydrogen peroxide when cultured in 48. 48 h.

Table 23 capability of Lactobacillus salivarius to produce hydrogen peroxide

Microorganism	Hydrogen peroxide production score-48 h
		Lactobacillus salivarius Lvagi-34	1
Lactobacillus salivarius Lvagi-35	1
		Lactobacillus salivarius Lvagi-42	1

Example 6 Gardnerella vaginalis inhibition test

After the lactobacillus mucilaginosus is activated, 0.1 mL bacteria liquid is taken and evenly mixed with a melted MRS solid culture medium, poured into a 6 cm plate, after complete solidification, the plate is cultivated at 37 ℃ for 48 h, and the plate is taken out, and a puncher with the inner diameter of 6 mm is used for punching on an agar culture medium to obtain bacteria cakes; after activation and transfer of gardnerella vaginalis (purchased from Beijing North Injury Biotechnology institute, BNCC 337545), 10 was taken with 10-fold gradient using sterile PBS buffer ^-1 Mixing the diluent 0.5 mL with 5.25 mL solid culture medium containing 5% horse serum and melted BHI, pouring into 9 cm plate, after complete solidification, putting lactobacillus mucilaginosus bacterial cake on the surface of BHI solid culture medium, symmetrically putting 4 bacterial cakes in each plate, arranging 2 parallel bacteria in each strain, putting into anaerobic sealing tank, adding anaerobic gas producing bag, culturing the plate in normal position for 48 h, measuring the size of the bacteriostasis zone by vernier caliper, and calculating average value.

As shown in Table 3, lactobacillus salivarius Lvagi-34 was effective in inhibiting the growth of Gardnerella. Under the same experimental conditions, the antibacterial capacity of the Lvagi-34 is stronger than that of the control strains Lvagi-35 and Lvagi-42 of the same genus.

TABLE 3 Gardnerella vaginalis inhibition ability

Microorganism	Diameter (mm) -average value of inhibition zone
		Lactobacillus salivarius Lvagi-34	9.18
Lactobacillus salivarius Lvagi-35	8.59
		Lactobacillus salivarius Lvagi-42	8.66

EXAMPLE 7 hemolysis experiment

The deposited lactobacillus salivarius lvagei-34 was inoculated into 5 mL of MRS broth medium at an inoculum size of 10% with enterococcus faecalis (beta hemolysis, CICC23658, purchased from the chinese industrial microorganism strain deposit management center) as a positive control and a blank medium as a negative control. All strains were anaerobically cultured in MRS broth culture medium at 37℃for 12 h to give activated strains. 2.5. Mu.L of each activated strain was inoculated onto Columbia blood plates (Shanghai Marjia biotechnology Co., ltd.) and 3 groups each were placed in parallel and observed after anaerobic culture at 37℃for 48 h.

The results show that: the colony of the positive control strain forms a completely transparent hemolytic ring with obvious limit, which is beta hemolysis; the medium surrounding the colony of L.uromyxobacteria Lvagi-34 was unchanged and was gamma-hemolyzed, i.e.not hemolyzed.

In conclusion, the lactobacillus salivarius Lvagi-34 can produce lactic acid and resist the acidic environment, and can adapt to and maintain the acidic environment of female genital tracts; the bacteria can also produce a certain amount of hydrogen peroxide, and the hydrogen peroxide is a self-cleaning substance with antagonism among bacteria, can inhibit harmful bacteria in genital tracts and promote the growth of beneficial bacteria; the gardnerella vaginalis has a good effect on inhibiting gardnerella vaginalis and can be used for preventing and/or treating diseases related to gardnerella vaginalis.

Claims

1. Lactobacillus salivarius with urinary tract mucusLimosilactobacillus urinaemulieris) The strain is selected from lactobacillus helveticus Lvagi-34 with a preservation number of CCTCC NO: M20231491.

2. The method for culturing a strain of Lactobacillus salivarius according to claim 1, comprising inoculating Lactobacillus salivarius into a medium, and culturing the strain in a proliferation manner to obtain a strain of Lactobacillus salivarius.

3. The culture method according to claim 2, wherein the medium is an MRS medium.

4. A food, health product or pharmaceutical composition comprising the lactobacillus casei strain as claimed in claim 1 or the lactobacillus casei strain as obtained by the cultivation method as claimed in claim 2 as an active ingredient.

5. The composition of claim 4, wherein the active ingredient of the composition further comprises a compound selected from the group consisting of Lactobacillus crispatusLactobacillus casei) Lactobacillus jenseniiLactobacillus jensenii) Lactobacillus johnsoniiLactobacillus johnsonii) Lactobacillus delbrueckiiLactobacillus delbrueckii) Lactobacillus gasseri @ andLactobacillus gasseri) One or more of the following.

6. The composition of claim 4, wherein the lactobacillus salivarius is the only active ingredient.

7. The composition of claim 4, wherein the composition comprises 10 in a single formulation ⁶ ~10 ¹⁵ Urinary tract mucus lactobacillus of CFU.

8. The pharmaceutical composition of claim 4 for preparing medicines for inhibiting or killing gardnerella vaginalisGardnerella vaginalis) Is used in the products of the formula (I).